Adrenocortical carcinoma (ACC) is a deadly malignancy with survival <35% at 5 years and occurs across the lifespan afflicting both young and older male and female veterans. Current treatment options of surgery, mitotane and chemotherapy are inadequate. The mechanisms underlying ACC tumorigenesis are poorly understood. Patients with genetic syndromes have an increased risk of ACC; however, the majority of cases are sporadic. To date, no pathway has been successfully targeted in ACC in clinical trials. Lack of progress in the field has been attributable to the lack of cell and animal models of ACC. Until recently, only one ACC tumor cell line and no animal models were available. We have approached this unmet need by the development of 4 new human ACC cell lines and the first patient derived xenograft (PDX) animal models (N=5) with a strategy to use these pre-clinical models to test bioinformatically identified targets for novel therapeutics. Our overarching goal is to understand the diverse molecular pathogenesis of human ACC and identify novel targets to test in our new cell and animal models. We analyzed multiple publically available genomic databases for candidate genes that are dysregulated and noted that ACC tumors harbored a consistent dysregulation of cell cycle control and DNA damage pathway constituents. We identified PBK (PDZ binding kinase) as the highest dysregulated candidate. PBK has been identified as a master regulator of mitotic proteins and is critical for cytokinesis. Its downstream effectors are cell specific. Although low or undetectable in most normal human tissues, it is overexpressed in diverse cancers including breast, colon, lung, prostate and lymphomas. Our preliminary data show that PBK controls the tumorigenic phenotype in ACC and that targeting PBK with either shPBK or the PBK inhibitor, HITOPK032, reduced cell proliferation and tumorigenic growth in our pre-clinical models. This application will dissect the mechanisms by which PBK inhibition or targeting exert their anti- tumorigenic effects. In addition, we propose to test the effects of PBK targeting in combination with Mitotane therapy, the only FDA approved drug for adrenal cancer. These studies will test the hypothesis that disruption of specific components of cell the cycle in conjunction with Mitotane, a known ER stress inducer, will enhance the anti-tumorigenic responses. Our novel ACC cell lines and PDX models provide a unique opportunity to advance the understanding of the diverse mechanisms of adrenal cancer tumorigenesis as well as test for the first time relevant ACC targets that will allow translation to our patients in the clinic. Our studies will fill an unmet need in the area of endocrine tumorigenesis that afflicts veterans of all ages and shed light into the biology of this poorly studied cancer. In addition, the data will provide insight into other cancers with these mitotic kinase drivers which affect veterans every day.